PC4 is a human positive cofactor for RNA polymerase II to mediate activator-dependent transcription through the interactions with both activation domains and the basal transcription machinery. We have found that the function of PC4 is modulated by phosphorylation. Both protein-protein interaction and in vitro transcription assays demonstrate that only unphosphorylated PC4 is functionally active. Although in vitro phosphorylation experiments indicate that recombinant PC4 can be phosphorylated by casein kinase II and protein kinase C, the results from mutation analysis and determination of molecular masses of phosphorylated and dephosphorylated forms of natural PC4 by mass spectrometry suggest that PC4 is hyperphosphorylated mainly by casein kinase II in vivo in the first serine-rich region. These observations demonstrate a first example of transcriptional cofactor negatively regulated by CKII phosphorylation. A paper describing this work has been published in PNAS. Subsequent to the publication of the paper, we further refined our phorphorylation site mapping techniques utilizing the MALDI-Ion trap mass spectrometer.